We propose to develop a more sensitive, specific multiplex serologic assay for the diagnosis of Lyme disease (LD). LD is the most common vector borne infectious disease in the United States, and as such it is a significant public health concern. The disease affects multiple organ systems including musculoskeletal, skin and nervous system, and is included in the differential diagnosis of multiple diseases. In the absence of Erythema migrans, the classic skin lesion of early LD, the diagnosis is established by the detection of an antibody response to Borrelia burgdorferi (Bb) in patients with objective findings suggestive of the disease. Prompt diagnosis is important because early treatment of LD limits or prevents serious damage to the systems affected. Current serodiagnostics lack sensitivity in early disease. We laid the ground work for a new generation of seroassays by mapping and defining the specific linear B cell epitopes of key Bb antigens expressed in early infection. We identified specific epitopes from 19 of antigens that are suited for use in multi- peptide-based assays. In collaboration with Bio-Rad Laboratories, we will develop a highly sensitive and specific Luminex(r) LD serodiagnostic utilizing multiple peptides containing specific linear epitopes key Bb antigens. Luminex(r) X-Map is becoming a standard technology in most large clinical diagnostic labs. Bio-Rad's BioPlex 2200 is currently used at over 200 locations in the US, including commercial clinical labs such as Quest Laboratories and LabCorp, large medical groups, such as the Mayo and Cleveland Clinics, and many University-based medical centers. Our novel and highly innovative approach will fill the void in LD diagnostics, especially in early LD, and will provide superior specificity and sensitivity compared to current assays in all phases of LD. In addition, our collaboration with Bio-Rad Laboratories offers a clear cost effective path to the commercialization of this much needed technology.